1. Field of the Invention
The present invention relates to a method for analyzing the phyletic lineage of scallop, and particularly to a method for analyzing such lineage by sequencing a non-coding region found in the nucleotide base sequence of scallop""s mitochondrial DNA (mtDNA).
2. Description of Prior Art
In the past, scallops have been caught and produced directly from the natural fishing grounds, but the recent increasing demands have led to artificial culturing of scallops for mass production. As a consequence thereof, certain artificial techniques have gone on to control the seeding nursery of seed scallops, which however raises a negative aspect to adversely affect the genetic structure of whole organisms, not to mention the scallops. Such artificial seeding and culture control will result in decreasing the pools of genes and diminishing the variety of phyletic lineages of scallops. This means that, in the near future, a population of particular scallops will reduce its variety of lineages and will not adapt themselves to variable changes in the surrounding circumstances to leave their offsprings, causing unexpected grave consequences. Hence, it is now required that the specific genetic structure of scallops be analyzed to determine their DNA polymorphism or hypervariable nucleotide sequence regions which are key factors to save varied lineages of scallops.
In particular, the Japanese scallops (Patinopecten yessoensis, which widely inhabit the cold coasts of the northern islands of Japan, the northern part of the Korean Peninsula, Sakhalin and the Kuril islands, are one of the most attractive scallops for food products and food processing. To retain the widest possible phyletic lineages of such Japanese scallops is also greatly required in view of the above-discussed artificially culturing problems.
Conventional methods for analyzing the genetic structure of one scallop population has been based on the use of isozymes or allozymes. But, such enzyme method has been found poor in sensitivity and insufficient as a tool to specifically determine and classify the lineage of each Japanese scallop population in a particular local marine area of Japan. Further, the problem of that conventional method is that it requires a great amount of samples to be analyzed, and time-consuming, inefficient steps of analyzing procedures.
In addition, there has been no report which completely analyzes the genetic characteristics of each different Japanese scallop population as well as the specific base sequence of mtDNA of the Japanese scallop. Thus, no data has been made available, which is necessary to keep the lineage of the Japanese scallops.
In view of the above-stated shortcomings, it is a primary purpose of the present invention to provide a method which assures to analyze a phyletic lineage of scallop with high precision and reliability.
For that purpose, a method in accordance with the present invention basically comprises the steps of sequencing a mitochondrial DNA (mtDNA) of the scallop to determine a nucleotide base sequence of the mtDNA, wherein the nucleotide base sequence includes a non-coding region containing a nucleotide base substitution locus which shows a sequence polymorphism indicative of a particular lineage of the scallop.
In one aspect of the present invention, the method may comprise the steps of: extracting a total DNA as a template DNA from an adductor muscle of the scallop; amplifying mtDNA from the template DNA under polymerase chain reaction, using a suitable primer set designed on the basis of nucleotide base sequences conserved among known shellfish mitochondrial 16S rRNA genes and 12S rRNA genes; sequencing the thus-amplified mtDNA while determining a non-coding region therein; thereafter, sequencing the non-coding region, using suitable primers designed on the basis of the determined nucleotide base sequences of the mtDNA; and locating a nucleotide sequence substitution locus in the non-coding region, thereby determining a particular lineage of the scallop.
Japanese scallop, Patinopecten yessoensis, is analyzed as to the nucleotide base sequence of mitochondrial DNA thereof, with the afore-said suitable primer set comprising Pyso 16S BF primer (5xe2x80x2-CGGCGMGCCAGGTCAGTTTCTATC-3xe2x80x2) (SEQ ID NO: 3) and Pyso 12S BR primer (5xe2x80x2-AGAGCGACGGGCGATGTGTACAC-3xe2x80x2), (SE ID NO:8) while locating a non-coding region in the base sequence, and then, the non-coding region of the mtDNA is sequenced, with the afore-said suitable primers comprising Pyso 16S BF primer (5xe2x80x2-CGGCGMGCCAGGTCAGTTTCTATC-3xe2x80x2) (SEQ ID NO:3), Pyso NcR primer (5xe2x80x2-AGGTAACCAGAACCAAACTACC-3xe2x80x2) (SEQ ID NO:10) and Pyso 12S AR primer (5xe2x80x2-ACTGCTGGCACCTGGTTGGA-3xe2x80x2) (SEQ ID NO:6).
In another aspect of the present invention, plural different amplified mtDNAs of different scallop samples, obtained by the foregoing polymerase chain reaction using the afore-said suitable primer set, may be grouped into different groups each corresponding to respective different scallop populations, and each of those amplified mtDNAs is sequenced while determining a non-coding region therein. Then, the non-coding region is sequenced, using the afore-said suitable primers, so that a nucleotide sequence substitution locus is located in the non-coding region with respect to each of the different groups, thereby determining sequence polymorphism in each of the groups, the sequence polymorphism being indicative of a particular lineage of each of the scallop, so as to classify the different scallops into particular lineages. In those steps, there may be included the steps of: comparing the nucleotide base sequence substitution loci of the different scallop samples with one another; selecting particular scallop samples from the different scallop samples, which particular samples are identical in nucleotide base sequence substitution loci to one another; obtaining a high population frequency among such particular samples; and determining identity of ancestor among the particular samples on the basis of the high population frequency so as to find identical ancestors among the different scallop populations.
In still another aspect of the invention, for high efficient analysis of plural scallop samples, after the amplification of mtDNAs of plural scallop samples, the non-coding regions of the mtDNAs may be directly sequenced, using the Pyso 16S BF, Pyso NcR and Pyso 12S AR primers, to locate nucleotide sequence substitution loci in the non-coding regions for quick analysis of lineage of each of the plural scallop samples on the different scallop population basis.
Another various advantages and features of the present invention will become apparent from reading of the descriptions hereinafter, with reference to the annexed drawings.